Alum is the only adjuvant approved for routine use in human vaccination although the basis for its adjuvanticity remains poorly understood. Nod-Like receptors (NLR) are pattern recognition receptors that detect microbial products and "danger signals" situated in the cytoplasm and trigger activation of the inflammasome, a multiprotein complex that regulates secretion of cytokines belonging to the IL-1 family (IL-12, IL-18, and IL-33). We have recently demonstrated that alum induces secretion of IL-1 family cytokines by activating the NLR molecule NLRP3 and, more importantly, we showed that the adjuvant effect of alum is in good part dependent on NLRP3. Our results also show that other particulate adjuvants, such as QuilA and chitosan, are able to stimulate NLRP3 in vitro, suggesting that inflammasome activation may be a common mechanism of action of particulate adjuvants. Numerous studies documented the adjuvant activities of the IL-1 family cytokines, yet their involvement in alum adjuvanticity has not been established conclusively. In aim 1 we will test the hypothesis that the NLRP3-inflammasome and the IL-1 family cytokines mediate the adjuvanticity of alum and other particulate adjuvants. Recently published results also suggest that some of alum's effects may be mediated by release of uric acid or generation of reactive oxygen species (ROS). Both alum and uric acid activate the NLRP3-inflammasome, yet they differ substantially in the biological effects they trigger, suggesting they may differentially activate additional inflammasome-independent pathways. The existence of these pathways, which may contribute to alum's adjuvanticity, is supported by the residual immune response of NLRP3-/- mice to alum vaccination. In aim 2 we will test the role of Syk kinase, oxidative stress, and uric acid in alum's adjuvanticity. Our most recent results show that 7BIO, a necrosis-inducing indirubin derivative, activate the NLRP3-inflammasome and possess adjuvant properties. Necrotic cells are known to release danger signals, such as the HMGB1 protein, that possess adjuvant ability. In aim 3 we want to test the hypothesis that inclusion of 7BIO or HMGB1 into alum-based vaccines can improve the quality and extent of the resulting immune response by inducing Th1 responses. The long-term goal of our studies is to understand at the molecular level the mechanism of action of alum and other particulate adjuvants and to improve their immunostimulatory spectrum. PUBLIC HEALTH RELEVANCE: Alum is the only adjuvant approved for routine use in human vaccination although the basis for its immunostimulatory activity remains poorly understood. The studies described in this grant proposal will increase our understanding of the mechanism of action of alum. A deeper understanding of the mechanisms that determine the immunostimulatory properties of adjuvants is a prerequisite for the rational design of more sophisticated vaccines, which remain the most promising strategy to fight infectious diseases in both industrialized and developing countries.